IL-2 regulation of tyrosine kinase activity is mediated through the p70-75 beta-subunit of the IL-2 receptor

The stimulation of activated human T lymphocytes with IL-2 results in increased tyrosine kinase activity. IL-2 treatment of Tac+ T cells stimulates the rapid phosphorylation of multiple protein substrates at M of 116, 100, 92, 70 to 75, 60, 56, 55, 33, and 32 kDa. Phosphorylation on tyrosine residues was detected by immunoaffinity purification of protein substrates with Sepharose linked antiphosphotyrosine mAb, 1G2. Although phorbol ester stimulated serine phosphorylation of the IL-2R alpha (p55) subunit recognized by alpha TAC mAb, IL-2 did not stimulate any detectable phosphorylation of IL-2R alpha or associated coimmune precipitated proteins. In fact, the tyrosine phosphorylated proteins did not coprecipitate with alpha Tac antibody and similar phosphoproteins were stimulated by IL-2 in IL-2R alpha- human large granular lymphocytes which express only the 70 to 75 kDa IL-2R beta subunit of the high affinity IL-2R. Anti-Tac mAb could inhibit IL-2-stimulated tyrosine phosphorylation in activated T cells, which express both IL-2R subunits that together form the high affinity receptor complex, but not in large granular lymphocytes expressing only the IL-2R beta subunit. The data suggest that IL-2 stimulation of tyrosine kinase activities requires only the IL-2R beta subunit.     

Cdc42 promotes G1 progression through p70 S6 kinase-mediated induction of cyclin E expression

The Rho family GTPase Cdc42 is recognized for its role in cellular proliferation and transformation. However, the mechanism by which it promotes cell cycle progression has remained undefined. Using an inducible expression system, we show that constitutively active Cdc42 (Cdc42V12) is sufficient by itself to induce anchorage-independent but not mitogen-independent growth in NIH3T3 cells. However, Cdc42V12 markedly accelerates activation of cyclin E-Cdk2 in response to mitogen. These effects were highly specific, as the kinetics of cyclin D-Cdk4 activation was unaltered. Cdc42V12 promotes Cdk2 activation by selectively inducing cyclin E expression without affecting other regulatory proteins such as the p27 Cdk inhibitor or Cdc25A. Furthermore, Cdc42V12 was able to activate a reporter gene driven by the cyclin E promoter in the absence of exogenous mitogen or adhesion. Cyclin E induction was sensitive to rapamycin but not inhibitors of mitogen-activated protein kinases, implicating p70 S6 kinase (p70S6k) as the relevant mediator. Consistent with this notion, wild type and constitutively active alleles of p70S6k were sufficient to activate the cyclin E promoter. In sum, these studies provide novel insights into the mechanism by which Cdc42 promotes G1 progression.     

The overlooked greatwall: a new perspective on mitotic control

The role of the dual specificity protein phosphatase, Cdc25, in activating the cyclin-dependent kinase-cyclin B complex (Cdk1-CycB) by overcoming the inhibitory Wee1 kinase is a long-established principle for mitotic entry. Recently, however, evidence has emerged of a regulatory network that facilitates Cdk1-CycB activity by inhibiting the form of protein phosphatase 2A having a B55 regulatory subunit (PP2A-B55). Here, I review the genetic and biochemical evidence for Greatwall kinase and its substrate Endosulphine as the key components of this previously obscure regulatory network. Not only is the inhibition of PP2A-B55 by phospho-endosulphine required to prevent dephosphorylation of Cdk1-CycB substrates until mitotic exit, but it is also required to promote Cdc25 activity and inhibit Wee1 at mitotic entry. I discuss how these alternating states of preferential PP2A-B55 or Cdk1-CycB activity can have an impact upon the regulation of Polo kinase and its ability to bind different partner proteins as mitosis progresses.     

Abl deregulates Cdk5 kinase activity and subcellular localization in Drosophila neurodegeneration

Although Abl functions in mature neurons, work to date has not addressed Abl's role on Cdk5 in neurodegeneration. We found that beta-amyloid (Abeta42) initiated Abl kinase activity and that blockade of Abl kinase rescued both Drosophila and mammalian neuronal cells from cell death. We also found activated Abl kinase to be necessary for the binding, activation, and translocalization of Cdk5 in Drosophila neuronal cells. Conversion of p35 into p25 was not observed in Abeta42-triggered Drosophila neurodegeneration, suggesting that Cdk5 activation and protein translocalization can be p25-independent. Our genetic studies also showed that abl mutations repressed Abeta42-induced Cdk5 activity and neurodegeneration in Drosophila eyes. Although Abeta42 induced conversion of p35 to p25 in mammalian cells, it did not sufficiently induce Cdk5 activation when c-Abl kinase activity was suppressed. Therefore, we propose that Abl and p35/p25 cooperate in promoting Cdk5-pY15, which deregulates Cdk5 activity and subcellular localization in Abeta42-triggered neurodegeneration.     

Cloning of three novel neuronal Cdk5 activator binding proteins

Neuronal Cdc2-like kinase (Nclk) is involved in the regulation of neuronal differentiation and neuro-cytoskeleton dynamics. The active kinase consists of a catalytic subunit, Cdk5, and a 25 kDa activator protein (p25nck5a) derived from a 35 kDa neuronal-specific protein (p35nck5a). As an extension of our previous study (Qi, Z., Tang, D., Zhu, X., Fujita, D.J., Wang, J.H., 1998. Association of neurofilament proteins with neuronal Cdk5 activator. J. Biol. Chem. 270, 2329-2335), which showed that neurofilament is one of the p35nck5a-associated proteins, we now report the isolation of three other novel p35nck5a-associated proteins using the yeast two-hybrid screen. The full-length forms of these three novel proteins, designated C42, C48 and C53, have a molecular mass of 66, 24, and 57 kDa, respectively. Northern analysis indicates that these novel proteins are widely expressed in human tissues, including the heart, brain, skeletal muscle, placenta, lung, liver, kidney and pancreas. The bacterially expressed glutathione S-transferase (GST)-fusion forms of these three proteins were able to co-precipitate p35nck5a complexed with Cdk5 from insect cell lysate. Among these three proteins, only C48 and C53 can be phosphorylated by Nclk, suggesting that they may be the substrates of Nclk. Sequence homology searches have suggested that the C48 protein is marginally related to restin protein, whereas the C42 protein has homologues of unknown function in Caenorhabditis elegans and Arabidopsis thaliana.     

Cyclin‐dependent kinase 9 forms a complex with GATA4 and is involved in the differentiation of mouse ES cells into cardiomyocytes

The treatment of ES cells with trichostatin A (TSA), an HDAC inhibitor, induces the acetylation of GATA4 as well as histones, and facilitates their differentiation into cardiomyocytes. Recently, we demonstrated that cyclin‐dependent kinase 9 (Cdk9), a core component of positive elongation factor‐b, is a novel GATA4‐binding partner. The present study examined whether Cdk9 forms a complex with GATA4 in mouse ES cells and is involved in their differentiation into cardiomyocytes. Mouse ES cells and Nkx2.5/GFP ES cells, in which green fluorescent protein (GFP) is expressed under the control of the cardiac‐specific Nkx2.5 promoter, were induced to differentiate on feeder‐free gelatin‐coated plates. Immunoprecipitation/Western blotting in nuclear extracts from mouse ES cells demonstrated that Cdk9 as well as cyclin T1 interact with GATA4 during myocardial differentiation. TSA treatment increased Nkx2.5/GFP‐positive cells and endogenous mRNA levels of Nkx2.5 and atrial natriuretic factor. To determine the role of Cdk9 in myocardial cell differentiation, we examined the effects of a dominant‐negative form of Cdk9 (DN‐Cdk9), which loses its kinase activity, and a Cdk9 kinase inhibitor, 5,6‐dichloro‐1‐β‐ribofuranosyl‐benzimidazole (DRB) on TSA‐induced myocardial cell differentiation. The introduction of the DN‐Cdk9 inhibited TSA‐induced increase in GFP expression in Nkx2.5/GFP ES cells. The administration of DRB into ES cells significantly inhibited TSA‐induced increase of endogenous Nkx2.5 mRNA levels in ES cells as well as GFP expression in Nkx2.5/GFP ES cells. These findings demonstrate that Cdk9 is involved in the differentiation of mouse ES cells into cardiomyocytes by interacting with GATA4. J. Cell. Physiol. 226: 248–254, 2010. © 2010 Wiley‐Liss, Inc.     

A major role of PKC theta and NFkappaB in the regulation of hTERT in human T lymphocytes

Expression of the telomerase catalytic subunit (TERT) is the rate-limiting determinant of telomerase activity in most human cells. In this work, we examined the participation of protein kinase C (PKC) in the regulation of hTERT expression in human T lymphocytes. Transient expression assays using luciferase reporter plasmids containing hTERT promoter showed that overexpression of PKC θ, but not the other PKC isoforms, could activate the promoter activity of hTERT in resting T lymphocytes. Among the PKC θ-activated signalings, we presented evidence that the expression of hTERT is mediated through NFκB but not through MEK or c-Jun N-terminal kinase pathways. Analysis of the hTERT promoter occupancy in vivo using chromatin immunoprecipitation assays, however, did not detect an increased binding of NFκB to the hTERT promoter in the activated T cells, although an increased binding of cMyc and Sp1 was detected. Together with the observation that inhibition of NFκB eliminated the induction of cMyc in activated T cells, these results suggest that PKC θ-activated NFκB signaling regulates the expression of hTERT via cMyc in human T lymphocytes.     

Development and characterization of polyclonal antibodies against a conserved sequence in the catalytic domain of protein kinases.

Using a synthetic oligopeptide (CGGGTPEYLAPEGGK) crosslinked to keyhole limpet hemocyanin we have raised polyclonal rabbit antibodies against a 9 residue homologous region found in the catalytic domain of most protein kinases. These antibodies reacted during Western immunoblotting with cAMP dependent protein kinase catalytic subunit, phosphorylase kinase gamma subunit and calcium calmodulin dependent protein kinase II which have homologous sequences of GTPEYLAPE, GTPSYLAPE and GTPGYLSPE, respectively. Five other protein kinases did not react with anti-GTPEYLAPE antibodies during Western immunoblotting. Affinity-purified antibodies were able to detect as little as 50 ng of cAMP dependent protein kinase and 200 ng of Ca2+/calmodulin dependent protein kinase II. Immunoblotting of A431 cell plasma membrane vesicles indicated the presence of an approximately 55 kDa protein that contains the conserved sequence and is likely to be a protein kinase. Antibodies directed against conserved sequences present in protein kinases, or possibly other enzymes, may be useful in identifying previously uncharacterized enzymes at the protein level.